APOE and neuroinflammation as modulators of glia-neuron communication via extracellular vesicles: implications for Alzheimer's disease

细胞外小泡交换(EVS，指的是外体和微泡)作为一种新的机制已经出现 中枢神经系统中的细胞间通讯，但对这些细胞的生物学功能知之甚少 健康或疾病的转移。星形胶质细胞是中枢神经系统(CNS)最丰富的细胞类型之一， 分泌对神经元健康有益或有害的EV，这取决于它们的激活状态，因此暗示 星形胶质细胞衍生的电动汽车可以调节大脑对压力的韧性和脆弱性。有趣的是，我们最近 观察到，载脂蛋白E(APOE)，阿尔茨海默病(AD)最相关的遗传风险调节因子，可以 发现与从体外星形胶质细胞分离的EVS有关，星形胶质细胞是大脑中产生载脂蛋白E的主要细胞类型。而当 几十年来，载脂蛋白E一直被认为是神经细胞之间脂质的主要载体和淀粉样蛋白的强大伙伴 阿尔茨海默病中的β多肽(Apo4和Apo2)分别增加或减少淀粉样蛋白在脑内的积聚并与 更高的风险或对疾病的保护)，这一发现表明载脂蛋白E在调节 许多其他具有生物活性的化合物通过电动汽车，包括核酸。因此，本提案 假设星形胶质细胞和神经元之间的EV交换可以被不同的APOE调节 异构体，依赖于星形胶质细胞的反应性，至少部分地对一些非细胞自主效应负责。 衰老和阿尔茨海默氏症。为了研究这些令人兴奋的问题，我们将进一步表征 APOE与人脑星形胶质细胞来源的EVS进行APOE，分析从 静息或反应星形胶质细胞并监测体内和体外这些参数(APOE亚型和激活 星形胶质细胞的状态)可能最终影响EV神经元的摄取。重要的是，因为电动汽车是不能 很容易被常规显微镜观察到，因为所有类型的细胞都可以分泌和捕获EV，研究它们的 在体内的交流被证明是非常具有挑战性的。为了绕过这个问题，我们开发了一种新颖的记者系统 基于含有Cre重组酶mRNA的星形胶质细胞来源的EV的分泌(供体细胞型)和 Cre敏感报告基因在神经元中的表达(受体细胞类型)。使用这一创新的“开/关”记者系统， 我们将首次能够在活着的小鼠体内研究EV从星形胶质细胞到神经元的动态转移 通过多光子成像，确定这些过程是否受到每个APOE变体和 在脂多糖诱导的神经炎症后，随着年龄的增长或在阿尔茨海默病神经病变的背景下加重。 重要的是，我们还将能够从相同的生物细胞中分离出受体神经元和非受体神经元 并建立区分这两个种群的特定转录变化，最终将 这些发现与我们最初对从静息和反应性星形胶质细胞分离的EVS中检测到的RNA货物进行了筛选。 考虑到这一领域的知识匮乏，本项目将增进我们对生物学的理解 基于EV的细胞-细胞通讯在衰老或神经退行性变的背景下的相关性，最终打开了一篇新的 调查的途径。

Exchange of extracellular vesicles (EVs, referring to exosomes and microvesicles) has emerged as a novel mechanism of cell-cell communication in the central nervous system, but very little is known about the biological function of those transfers in health or disease. Astrocytes, one of the most abundant cell types of the central nervous system (CNS), secrete EVs that can be beneficial or detrimental to neuronal fitness depending on their activation state, thus implying that astrocyte-derived EVs can modulate brain resilience and vulnerability to stress. Interestingly, we have recently observed that apolipoprotein E (APOE), the most relevant genetic risk modulator of Alzheimer’s disease (AD), can be found in association with EVs isolated from astrocytes in vitro, the major cell type producing APOE in the brain. While APOE has been known for decades as the primary carrier of lipids between neural cells and a strong partner of Amyloid β peptides in AD (APOE4 and APOE2 respectively increasing or decreasing amyloid buildup in the brain and conferring higher risk or protection toward the disease), this discovery suggests a novel role of APOE in modulating the transfer of many other biologically active compounds through EVs, including nucleic acids. The present proposal therefore hypothesizes that the exchange of EVs between astrocytes and neurons can be differentially modulated by each APOE isoform, depends upon astroglial reactivity and is, at least in part, responsible for some non-cell autonomous effects in aging and Alzheimer’s disease. To investigate those exciting questions, we will further characterize the association of APOE with astrocyte-derived EVs from human brain samples, analyze the nucleic acid content of EVs released from resting or reactive astrocytes and monitor in vivo and in vitro, how those parameters (APOE isoforms and activation state of astrocytes) may eventually impact EV neuronal uptake. Importantly, because EVs are small particles that cannot be readily observed by conventional microscopy and because all cell types can secrete and capture EVs, studying their exchanges in vivo has proven very challenging. To circumvent this problem, we have developed a novel reporter system based on the secretion of astrocyte-derived EVs containing Cre recombinase mRNA (donor cell type) and on the expression of a Cre-sensitive reporter in neurons (recipient cell type). Using this innovative “ON/OFF” reporter system, we will be able, for the first time, to study the dynamic transfers of EVs from astrocytes to neurons in the living mouse brain by multiphoton-imaging, determine if those processes are modulated by the nature of each APOE variant and exacerbated after LPS-induced neuroinflammation, upon aging or in the context of AD neuropathological changes. Importantly, we will also be able to sort recipient neurons from non-recipient neurons from the same biological environment and establish the specific transcriptional changes that distinguish both populations, eventually correlating those findings with our initial screen of RNA cargos detected in EVs isolated from resting and reactive astrocytes. Considering the paucity of knowledge in the field, the current project will advance our understanding in the biological relevance of EV-based cell-cell communication in the context of aging or neurodegeneration, eventually opening novel avenues of investigation.